Yukuharu Kobayashi

A simple and rapid immunochromatographic test kit for rabies diagnosis

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine‐pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.

Development of an Immunochromatographic Test To Detect Antibodies against Recombinant Em18 for Diagnosis of Alveolar Echinococcosis

ABSTRACT An immunochromatographic test (ICT) for the rapid detection of antibodies to Echinococcus multilocularis was developed. The ICT showed a sensitivity of 94% and a specificity of 95.4%. High degrees of agreement were observed between the ICT and an enzyme-linked immunosorbent assay (κ = 0.93) and between the ICT and immunoblot analysis (κ = 0.97). It is expected that the ICT developed in this study will be useful for the serodiagnosis of alveolar echinococcosis.

Development and evaluation of a rapid neutralizing antibody test for rabies

The level of virus-neutralizing antibody, which plays a crucial role in the prevention of rabies, is determined by rabies virus (RABV) neutralizing test, which are time- and cost-consuming. In order to determine the level of neutralizing antibody in vaccinees, an easy and reliable method is needed. Based on the principle of immunochromatography, we developed a RAPINA (RAPId Neutralizing Antibody) test to determine the presence of neutralizing antibody in serum. In the RAPINA test, if neutralizing antibody equivalent to 0.5IU/ml of serum sample are mixed with an optimal amount of inactivated RABV (iRABV) and are completely absorbed by the virus, none of the iRABV can bind with monoclonal antibody that recognizes the iRABV glycoprotein (G) on the test strip. A total of 115 human sera samples were tested. The sensitivity, specificity and accuracy of the RAPINA test compared with rapid fluorescent focus inhibition test (RFFIT) as a standard test, were 88.7, 91.9 and 90.4%, respectively. The RAPINA test is a simple, safe and rapid method, which can be a substitute for neutralizing tests that use live viruses, cultured cells and fluorescence microscopy. This test might be useful for screening a large number of sera.

Broad-Spectrum Detection of H5 Subtype Influenza A Viruses with a New Fluorescent Immunochromatography System

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 103 pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10–100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.

Development of a Rapid Diagnostic Kit That Uses an Immunochromatographic Device To Detect Antibodies in Human Sparganosis

ABSTRACT A diagnostic kit using an immunochromatographic device was developed to replace the time-consuming immunodiagnostic methods for human sparganosis. The kit was found to be faster and easier to use than an enzyme-linked immunosorbent assay (ELISA) and showed higher sensitivity and specificity. It will be useful for the laboratory diagnosis of hospitalized cases of sparganosis.

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.

Fluorescent Immunochromatography for Rapid and Sensitive Typing of Seasonal Influenza Viruses

Lateral flow tests also known as Immunochromatography (IC) is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60%) and the limit of detection (LOD) is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC) for subtyping H5 influenza viruses (FLIC-H5). Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD) is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB). This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75), specificity 100% (54/54), Type B: sensitivity 100% (90/90), specificity 98.2% (54/55) in nasal swab samples) in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13h) than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.

Multi-colored immunochromatography using nanobeads for rapid and sensitive typing of seasonal influenza viruses

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza viruses because of its rapid turnaround and ease of use. Despite the usefulness of IC, the limit of detection of common IC kits is as high as 10(3)-10(4) plaque forming units (pfu) per reaction, resulting in their limited sensitivities. Early diagnosis within 24h would provide more appropriate timing of treatment. In this study, a multi-colored NanoAct™ bead IC was established to detect seasonal influenza viruses. This method has approximately 10-fold higher sensitivity than that of colloidal gold or colored latex bead IC assays, and does not require specific instruments. More notably, NanoAct™ bead IC can distinguish influenza A and B viruses from clinical samples with a straightforward readout composed of colored lines. Our results will provide new strategies for the diagnosis, treatment, and a chance to survey of influenza viruses in developing countries and in the field research.